Biochem I Lecture 15: Irreversible Inhibition & Enzyme Efficiency

what does a lower concentration cause?

faster turn-out

what is [E]total?

everything

is ES -> E + P reversible or irreversible?

it's irreversible

- [P]o=0

- [S] > [E]total

- [E]total is constant

what is Vmax?

how quickly the product is being made with respect to the concentration of enzyme that is available

what is kcat?

- the turnover number- number of substrate molecules converted to product per enzyme molecule per unit of time when E is saturated

- k2 = kcat

what values does kcat range from?

less than 1/sec to many millions/sec

catalytic efficiency

kcat/km

- how perfect the enzyme is

- second order rate constant

- measures how well the enzyme performs when S is low

what is the upper limit of kcat/km?

diffusion limit- rate at which E and S diffuse together (usually super fast)

what is the perfect catalytic efficiency?

10^8-10^9

Ks

the dissociation constant of E and S

Km

SUBSTRATE CONCENTRATION AT 1/2 VMAX

DO NOT FORGET THIS!!!!

kcat

k2 when M-M applies (turnover rate)

kcat/km

catalytic efficiency (second order rxn- concentration AND time)

when [S] > Km

v approaches Vmax very quickly

when [S] < Km

rate follows first order kinetics (linear)

lineweaver-burk equation

1/v = (Km/Vmax)(1/[S]) + 1/Vmax

types of enzyme inhibition

reversible and irreversible

reversible inhibitors are

- non-covalent associate/dissociate

- competitive, noncompetitive (pure or mixed), or uncompetitive

irreversible inhibitors are

covalent bonds with side chains or other groups

- suicide substrates- gave themselves up to kill enzyme

inhibitor

any ligand, natural or synthetic, that decreased the velocity of an enzyme-catalyzed rxn

effect of inhibitor

change in Km and/or Vmax

- apparent Km or Vmax values in presence of inhibitor

how does the inhibitor change Km and Vmax values?

reversible binding to one or more enzyme present during the rxn

in competitive inhibitors,

- substrate and inhibitor compete for the SAME site

- they are often structurally similar

- IES does not form, either EI or ES

- EI does NOT produce P

- reduces the production of ES which slows down the turnover rate

diagnostics of competitive inhibitors

- vmax unaffected

- km increased

- high [S] will overcome I

what does dihydropteroate synthase do?

it synthesizes tetrahydrofolate in bacteria using PABA as a substrate

what is tetrahydrofolate?

a coenzyme in metabolism of amino acids and nucleic acids

what is methanol converted into and by what?

the liver alcohol dehydrogenase converts it into formaldehyde which leads to blindness and death

what does ethanol compete with?

methanol for binding to alcohol dehydrogenase in order to slow the production of formaldehyde

how is methanol secreted?

urine

non-competitive inhibitors

- I does NOT bind to same site as S

- I will bind to E or ES, so IES is possible

- can be pure (rare) or mixed

what is KI

inhibition constant when I binds to E

- KI=K'I in pure non-competitive inhibitors

when is K'I?

inhibition constant when I binds to ES

in pure noncompetitive inhibition, the binding of I does not influence what?

binding of S (it's quite rare since there's a loss of affinity)

does KI equal K'I in mixed noncompetitive inhibition?

NO

diagnostics of pure noncompetitive inhibition

- vmax reduced (shifted upwards)

- km is unchanged

- as if [E] was reduced

in mixed noncompetitive inhibition, the binding of I influences what?

the binding of S, this is common

diagnostics of mixed noncompetitive inhibition

- Vmax is reduced

- km is increased or decreased

when is Km increased in mixed noncompetitive enzyme inhibitors?

when KI < K'I

when is Km decreased in mixed noncompetitive enzyme inhibitors?

KI > K'I

uncompetitive inhibitors

- I combines with ES (not E)

- doesn't bind to enzyme until ES is formed

- reduces k2

uncompetitive inhibitors diagnostics

- vmax is decreased

- km is decreased

- km/vmax or gradient does not change

irreversible inhibition

- I combines with E irreversibly

- net effect of loss of E

- time-dependent decrease in enzymatic activity as EI forms

- dialysis or dilution will NOT remove I

- suicide substrates

in irreversible inhibition, I resembles what?

it resembles S and the E acts on it. However, instead of producing the product, I is covalently attached to a functional group in the active site

what is an example of an irreversible inhibitor?

penicillin

- covalently attaches to a serine residue in the active site- glycoprotein peptidase

- cells will die by osmotic lysis

organophosphate, parathion insecticides, and sarin nerve gas

these are examples of irreversible inhibitors. They inhibit the breakdown of neurotransmitter acetylcholine by acetylcholinesterase. This inhibits anything from occurring